Signals which recruit neutrophils to sites of inflammation are being actively studied, yet neither the initiating stimuli nor the final effector molecules are known with certainty. In this grant proposal we hypothesize that early formation of nontoxic levels of reactive oxygen intermediates (ROI) by resident cells in the tissue (endothelial cells, fibroblasts) induce interleukin 8 (IL-8) gene expression. IL-8, in turn recruits neutrophils to the site of inflammation. Multiple pathophysiologic conditions will cause the formation of ROI, and the normal cells endogenous to the tissues are capable of ROI synthesis. This hypothesis does not contradict previous work that ROI may serve as causative factors of tissue injury, but expands their role by suggesting that they do not function solely as end stage effector molecules, but also as mediators regulating cytokine cascades. While many chemoattractants have been described, IL-8 possesses unique properties allowing it to play an critical role in the recruitment and activation of neutrophils. We intend to carefully dissect the interactions between ROI and induction of IL-8 gene expression. Specific aim I will examine mechanisms accounting for the unusual stability of IL-8 at the mRNA level. Specific aim II will use primary cell cultures to test the central hypothesis that generation of ROI leads to IL-8 gene expression. This will be accomplished by a combined approach of l) stimulating cells in the presence of ROI scavengers or specific enzyme inhibitors and documenting decreased IL-8, and 2) directly inducing ROI and showing upregulation of IL-8. Endogenous, intracellular levels of oxygen metabolizing enzymes will be modulated by incubating the cells with antisense oligonucleotide probes to the mRNAs. This work will be conducted with endothelial cells, fibroblasts, and monocytes, to clearly demonstrate that the endogenous cellular components both synthesize ROI, and generate an IL-8 response. Experiments will be initiated with cell lines, and results verified with primary cultures. Careful, kinetic studies will be performed, where the addition of antioxidants will be delayed. These experiments will be done to distinguish the role of ROI in prevention of IL-8 induction from the role of ROI in tissue injury. Specific aim III will ascertain which specific ROI are responsible for the induction of IL-8. After stimulation, ROI will be measured to document their upregulation. To further define the ROI, enzyme inhibitors and ROI scavengers will be employed. The data from these approaches to specific aim III will be compiled to arrive at a conclusion, since none by itself would be persuasive. Successful completion of all of these studies will advance our understanding of the basic mechanisms of inflammation. Delineation of the regulatory events in acute inflammation leading to neutrophil recruitment will provide insights that may evolve into better, more directed therapeutic interventions.